Conselho Nacional de Desenvolvimento Científico e Tecnológico / In Southern Brazil, the plant Campomanesia xanthocarpa Berg. (Myrtaceae), popularly known as guavirova , has been empirically used for its potential effect in reducing blood cholesterol levels. Since there are no scientific data confirming its popular use, the aim of the present study was to investigate the effect of C. xanthocarpa on biochemical, hematological, anthropometrical and oxidative stress parameters in hypercholesterolemic patients. Thirty three patients were selected according to total cholesterol (TC) levels: 200-240 mg/dL, undesirable level (UL), and >240 mg/dL, hypercholesterolemic level (HL). UL or HL patients were randomly divided into control group (CG), which received placebo capsules, and experimental group 250 (EG 250) or 500 (EG 500), which received either 250 or 500 mg of encapsulated C. xanthocarpa. All groups received a cholesterol restriction diet and capsules once a day. The biochemical (TC, triglycerides, HDL, LDL and VLDL), hematological (hematocrit and hemoglobin), anthropometrical (weight and abdominal circumference) and oxidative stress (protein carbonyl) parameters were measured before, 45 and 90 days after the treatment started. There was no alteration on biochemical, hematological, anthropometric or oxidative stress parameters in UL patients of all groups. However, a significant decrease in TC and LDL levels was observed in HL patients from EG 500 group (reduction of 28±3 and 45±4% to levels before treatment) in relation to CG group patients (reduction of 12±2 and 29±4%). Moreover, a significant reduction in oxidative stress was observed in HL patients of EG 250 (51±12%) and EG 500 groups (34±18%) when compared to levels before treatment. A positive correlation between plasma oxidative stress PC and TC levels was observed. Finally, was demonstrated that C. xanthocarpa extract possesses anti-oxidant properties and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitory activity in vitro. Confirming its popular use, the treatment with C. xanthocarpa encapsulated reduced blood TC and LDL levels in hypercholesterolemic patients. Besides its effect on cholesterol levels, this plant reduced the oxidative stress in plasma of hypercholesterolemic patients as well. / No Sul do Brasil, a planta Campomanesia xanthocarpa Berg. (Myrtaceae), popularmente conhecida como guavirova , tem sido empiricamente usada por seu efeito potencial em reduzir os níveis de colesterol sanguíneo. Uma vez que não há dados científicos confirmando seu uso popular, o alvo do presente estudo foi investigar os efeitos da C. xanthocarpa nos parâmetros bioquímicos, hematológicos, antropométricos e de estresse oxidativo em pacientes hipercolesterolêmicos. Trinta e três pacientes foram selecionados de acordo com os níveis de colesterol total (CT): 200-240 mg/dL, níveis indesejáveis (NI), e > 240 mg/dL, níveis hipercolesterolêmicos (NH). Os pacientes NI e NH foram randomicamente divididos em grupo controle (GC), que recebeu cápsulas placebo, e grupo experimental 250 (GE 250) ou 500 (GE 500), que recebeu 250 ou 500 mg de C. xanthocarpa encapsulada. Todos os grupos receberam uma dieta com restrição a colesterol e cápsulas diariamente. Os parâmetros bioquímicos (CT, triacilgliceróis, HDL, LDL e VLDL), hematológicos (hematócrito e hemoglobina), antropométricos (peso e circunferência abdominal) e de estresse oxidativo (proteína carbonilada) foram mensurados antes, 45 e 90 dias depois do tratamento. Não houve nenhum alteração significativa nos parâmetros bioquímico, hematológico, antropométrico e de estresse oxidativo em pacientes NI de todos os grupos. Entretanto, uma redução significativa nos níveis de CT e LDL foi observada em pacientes NH do GE 500 (redução de 28±3 e 45±4% para os níveis antes do tratamento) em relação aos pacientes do GC (redução de 12±2 e 29±4%). Além disso, uma redução significante no estresse oxidativo foi observada em pacientes NH do GE 250 (51±12%) e GE 500 (34±18%) quando comparado com os níveis antes do tratamento. Uma correlação positiva entre os níveis de proteína carbonilada e CT foi observada. Finalmente, foi demonstrado que o extrato de C. xanthocarpa possui propriedade antioxidante e atividade inibitória da 3-hidroxi-3-metilglutaril coenzima A redutase in vitro. Confirmando seu uso popular, o tratamento com C. xanthocarpa reduziu os níveis de CT e LDL sanguíneos em pacientes hipercolesterolêmicos. Além dos seus efeitos nos níveis de colesterol, esta planta reduziu o estresse oxidativo no plasma de pacientes hipercolesterolêmicos.

Organic nitrates, such as nitroglycerine (NTG), are widely used for their potent vasodilator capacity in the management of coronary artery disease and heart failure. Unfortunately, their beneficial effect is rapidly lost due to the development of nitrate tolerance, which is translated by an impaired vasorelaxation to NTG and an increased oxidative stress production. Although the mechanisms of the development of nitrate tolerance are still not fully elucidated, much interest has been focused in treating nitrate-receiving patients together with other drugs in order to overcome the development of nitrate tolerance. The Nitric Oxide generating enzyme, eNOS, and the superoxide anion generating enzyme, NAD(P)H oxidase, have been suggested to play a role in the development of nitrate tolerance. The aim of this study was to analyse the underlying mechanism by which ramipril, an ACE inhibitor and rosuvastatin, a new molecule of the statin class, are able to protect against the development of nitrate tolerance in the aortas isolated from rats, wild-type (wt) and eNOS-/- mice. <p>These results show that ramipril as well as rosuvastatin are able to protect against the development of nitrate tolerance in the wt and eNOS-/- mice aortas suggesting that eNOS is not necessary for their protective effect. The aortas from nitrate tolerant rats and mice showed a significant increase in the NAD(P)H oxidase activation compared to the aortas from the control and from the co-treated ramipril+NTG or rosuvastatin+NTG animals. In line with these findings were the results obtained by RT-PCR analysis: the mRNA expression of the different subunits of the NAD(P)H oxidase, such as gp91phox, p22phox, were significantly decreased after rosuvastatin or ramipril treatment in wt and eNOS-/- mice aortas. Apocynin, the NAD(P)H oxidase inhibitor was also able to inhibit the development of nitrate tolerance in the rat and mouse aortas. <p>In conclusion, these results suggest that rosuvastatin and ramipril are able to protect against the development of nitrate tolerance by counteracting the nitrate-induced oxidative stress. The mechanism of protection involves a direct interaction with the NAD(P)H oxidase pathway and seems to be completely independent of the eNOS pathway. <p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

Pantothenate kinase (PanK) is an ubiquitous and essential enzyme that catalyzes the first step in the universal Coenzyme (CoA) biosynthesis pathway. In this step, pantothenate (Vitamin B5) is converted to 4′-phosphopantothenate, which subsequently forms CoA in four enzymatic steps. In bacteria, three types of PanK’s have been identified which exhibit wide variations in their distribution, mechanisms of regulation and affinity for substrates. Type I PanK is a key regulatory enzyme in the CoA biosynthesis pathway and its activity is feedback regulated by CoA and its thioesters. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on type I PanK from Mycobacterium tuberculosis (MtPanK) was initiated and the structure of this enzyme in complex with a CoA derivative has been reported earlier. To further elucidate the structural basis of the enzyme action of MtPanK, several crystal structures of the enzyme in complex with different ligands have been determined in the present study. In conjunction to this, solution studies on the enzyme were also carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion method was used for crystallization in all cases. The X-ray intensity data were collected using a MAR Research imaging plate system mounted on a Rigaku RU200 and Bruker-AXS Microstar Ultra II rotating anode X-ray generator. The data were processed using the HKL and MOSFLM and SCALA from the CCP4 suite. The structures were solved by the molecular replacement method using the program AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT and the refined structures were validated using PROCHECK and MOLPROBITY. Secondary structure was assigned using DSSP, structural superpositions were made using ALIGN and buried surface area was calculated using NACCESS. Solution studies on CoA binding and catalytic activity were carried out using Isothermal titration calorimetry (ITC). To start with, the crystal structures of the complexes of MtPanK were determined with (a) citrate, (b) the non-hydrolysable ATP analog AMPPCP and pantothenate (initiation complex), (c) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (end complex), (d) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (intermediate complex), (e) CoA, (f) ADP prepared by soaking and co-crystallization, which turned out to have identical structures and (g) ADP and pantothenate. Unlike in the case of the homologous E.coli enzyme (EcPanK), AMPPCP and ADP occupied different, though overlapping, locations in the respective complexes; the same was true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK was found to be substantially preformed while that of EcPanK exhibited considerable plasticity. The difference in the behavior of the E.coli and M.tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action and the changes in the locations of ligands exhibited by M.tuberculosis pantothenate kinase are remarkable and novel. The movement of ligands exhibited by MtPanK during enzyme action appeared to indicate that the binding site of the enzyme was less specific for a particular type of ligand than EcPanK. Kinetic measurements of enzyme activity showed that MtPanK had dual substrate specificity for ATP and GTP, unlike the enzyme from E.coli which showed a much higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes was provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate (2) GDP and phosphopantothenate (3) GDP (4) GDP and pantothenate (5) AMPPCP and (6) GMPPCP and the structures of the complexes of the two enzymes involving CoA and different adenyl nucleotides. The explanation was substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. Dual specificity of the type exhibited by this enzyme is rare and so are the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands and specificity. The crystal structures of MtPanK in binary complexes with nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP) provided insights about the natural location and conformation of nucleotides. In the absence of pantothenate, the NDP and the NTP bound with an extended conformation at the same site. In the presence of pantothenate, as seen in the initiation complexes, the NTP had a closed conformation and an altered location. However, the effect of the nucleotide on the conformation and the location of pantothenate were yet to be elucidated as the natural location of the ligand in MtPanK was not known. This lacuna was sought to be filled through X-ray analysis of the binary complexes of MtPanK with pantothenate and two of its derivatives, namely, pantothenol and N-nonyl pantothenamide (N9-Pan). These structures demonstrated that pantothenate, with a somewhat open conformation occupied a location similar to that occupied by phosphopantothenate in the “end” complexes, which was distinctly different from the location of pantothenate in “closed” conformation in the ternary “initiation” complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appeared to play a critical role in the movement of ligand that took place during enzyme action. The structure analysis of the binary complexes with the vitamin and its derivatives completed the description of the locations and conformations of nucleoside di and triphosphates and pantothenate in different binary and ternary complexes. These complexes provide snapshots of the course of action of MtPanK.

No description available.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 29, 2010). Vita. Thesis advisor: Grace Y. Sun. "May 2008" Includes bibliographical references.

La prévention des maladies cardiovasculaires constitue actuellement une approche capitale dans la diminution de la mortalité au sein de nos pays industrialisés. Tous les facteurs de risques étant associés à une dysfonction endothéliale, nous nous sommes intéressés à deux classes de médicaments dont l’action bénéfique se situe, du moins en partie, au niveau de l’endothélium vasculaire :les inhibiteurs de l’enzyme de conversion de l’angiotensine (IECA) et les inhibiteurs de l’hydroxy-3-méthyl-3-glutaryl-Coenzyme A (HMG-CoA) réductase (statines).<p> Le présent travail contribue à l’étude in vitro des effets protecteurs vasculaires de l’administration chronique, chez le rat, de deux statines (la pravastatine et l’atorvastatine) vis-à-vis de la toxicité aiguë des LDL humaines oxydées et vis-à-vis de la tolérance à la nitroglycérine. Une comparaison est menée par rapport au ramipril dans ces deux modèles expérimentaux.<p>Les effets de ces médicaments se manifestent au niveau vasculaire par une amélioration de la disponibilité du NO. Toutefois, dans nos modèles, des mécanismes singulièrement différents ont été identifiés entre les agents étudiés :alors que le ramipril engendre une augmentation de l’expression de la eNOS, enzyme synthétisant le NO, les statines permettent une meilleure disponibilité de ce radical par un mécanisme post-traductionnel. Outre cette action, elles semblent agir directement sur des enzymes oxydatives comme les NAD(P)H oxydases.<p>Une action antioxydante des statines pourrait expliquer tous les effets observés, ce qui n’est pas le cas pour le ramipril. Vu que le stress oxydatif intervient dans tous les facteurs de risques cardiovasculaires, diverses perspectives cliniques sont envisagées afin d’améliorer l’approche thérapeutique de la maladie athéroscléreuse.<p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

We now report an in depth analysis of the successful in vitro enzymatic synthesis of PHB utilizing the three-enzyme system from the bacteria Cupriavidus necator. Using HPLC methodology developed in this laboratory, and by adding each enzyme in a step-wise manner, we follow each individual stage in the three-enzyme route for PHB synthesis and delineate all stoichiometric relationships. We report the construction of the first metabolic model developed specifically for analyzing in vitro enzymatic PHB synthesis. We developed a hands-on student laboratory for culturing, producing, isolating, and purifying the bacterial biopolyesters PHB. We now report the first structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using MS, MS/MS, and homo- and hetero-nuclear NMR analyses.We describe HPLC methodology to separate the isomers of several iso-CoA-containing compounds and report the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl-transfer reactions. We describe a simple regioselective synthesis of iso-CoA from CoA. We also demonstrate a plausible mechanism, which accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. Herein we report that phenylaminoethyl selenide compounds protect DNA from peroxynitrite-mediated single-strand breaks. The mechanism of protection against peroxynitrite mediated DNA damage was investigated by HPLC. The chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. The unique chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. We report the development of novel CDB derivatives, which are selective COX-II inhibitors. A series of compounds were assayed with an in vitro colorimetric inhibitor screening and with a whole blood ELISA screening and the results indicate that MST is a selective inhibitor of COX-II.